Site-Specific Evolution of DNA Aptamers to Detect ADAR1p150 in Complex Biological Samples

Adenosine deaminase acting on RNA1 (ADAR1), particularly its interferon-inducible isoform ADAR1p150, is a key immune checkpoint in cancer and a promising prognostic biomarker. However, isoform-specific detection remains challenging. Here, we developed a sensitive detection platform for ADAR1p150 by selecting a high-affinity aptamer targeting its unique Zα domain using Blocker-SELEX, a strategy integrating structure-guided virtual screening and iterative sequence optimization based on affinity and competitive fluorescence polarization evaluation. The evolved aptamer, Zα_LS_11, bound the Zα domain with a dissociation constant (<i>K</i>_D) of 4.76 ± 2.91 nM. NMR titration and mutagenesis confirmed that Zα_LS_11 engages the target epitope, with the T191A mutation drastically reducing the affinity. The aptamer showed high specificity for ADAR1p150 over unrelated proteins and successfully captured endogenous ADAR1. We then constructed an aptamer-antibody sandwich assay, where ADAR1p150 is captured by the anti-ADAR1p150 antibody on magnetic beads and detected by an elongated aptamer (Zα_LS_20) via qPCR. This platform exhibited a linear detection range from 62.5 to 4000 ng/mL and reliably distinguished ADAR1p150 expression in cell lysates with or without IFNγ stimulation. Our work provides a robust, quantitative tool for monitoring ADAR1p150 dynamics, offering potential for assessing tumor immune status and prognosis.